Isolated ductal fluid sample

ABSTRACT

A sample for diagnosis of breast cancer can be prepared by isolating a ductal fluid sample from one duct of a breast of a patient. The isolated ductal fluid is not mixed with ductal fluid from any other duct of the breast. Generally the target duct is not spontaneously discharging. The isolated ductal fluid sample can be examined to determine the presence or absence of a marker associated with cancer or pre-cancer. An isolated ductal fluid sample not mixed with ductal fluid from any other duct of the breast permits identification of the duct which is diseased and provides increased sensitivity for existing diagnostic and analytic techniques.

CROSS-REFERENCES TO RELATED APPLICATIONS

This application is a continuation-in-part of application Ser. No.09/625,399 filed Jul. 26, 2000, which is a continuation-in-part ofapplication Ser. No. 09/502,404, filed on Feb. 10, 2000, which was acontinuation-in-part of application Ser. No. 09/313,463, filed on May17, 1999. This application is also a continuation-in-part of applicationSer. No. 09/473,510, filed on Dec. 28, 1999. This application alsoclaims the benefit under 37 CFR 1.78 of provisional application No.60/166,100 filed on Nov. 17, 1999. The full disclosures of each of theprior applications are incorporated herein by reference.

BACKGROUND OF THE INVENTION

For several decades significant members of the medical communitydedicated to studying breast cancer have believed and shown that thecytological analysis of cells retrieved from nipple discharge from thebreast milk ducts can provide valuable information for identifyingpatients at risk for breast cancer. Papanicolaou himself contributed tothe genesis of such a possibility of a “Pap” smear for breast cancer byanalyzing the cells contained in nipple discharge. See Papanicolaou etal, “Exfoliative Cytology of the Human Mammary Gland and Its Value inthe Diagnosis of Cancer and Other Diseases of the Breast” Cancer (1958)March/April 377-409. See also Petrakis, “Physiological, biochemical, andcytological aspects of nipple aspirate fluid”, Breast Cancer Researchand Treatment 1986; 8:7-19; Petrakis, “Studies on the epidemiology andnatural history of benign breast disease and breast cancer using nippleaspirate fluid” Cancer Epidemiology, Biomarkers and Prevention(January/February 1993) 2:3-10; Petrakis, “Nipple Aspirate Fluid inepidemiological studies of breast disease”, Epidemiologic Reviews (1993)15:188-195. More recently, markers have also been detected in nipplefluid. See Sauter et al, “Nipple aspirate fluid a promising non-invasivemethod to identify cellular markers of breast cancer risk”, BritishJournal of Cancer 76(4): 494-501 (1997). The detection of CEA in fluidsobtained by a nipple blot is described in Imayama et al. (1996) Cancer78: 1229-1234. Further, an intraductal aspiration method forcytodiagnosis in situations of sp ontaneous nipple discharge (Hou et al,Acta Cytologica 2000 v. 44:1029-1034) describes use of intraductalaspiration to collect specimens from spontaneously discharging ducts inorder to make a cytodiagnosis.

Breast cancer is believed to originate in the lining of a single breastmilk duct; and additionally the human breast is believed to contain from6 to 9 of these ducts. See Sartorius, JAMA 224 (6): 823-827 (1973).Sartorius describes use of hair-like single lumen catheters that areinserted into breast ducts using an operating microscope and the ductswere flushed with saline solution as described in Cassels, D March 20,1973, The Medical Post, article entitled “New tests may speed breastcancer detection”. After the fluid was infused, the catheter was removedbecause it was too small to collect the fluid, the breast was squeezedand fluid that oozed onto the nipple surface was removed from thesurface by a capillary tube. Similarly, Love and Barsky, “Breast-ductendoscopy to study stages of cancerous breast disease”, Lancet348(9033): 997-999, 1996 describes cannulating breast ducts with asingle lumen catheter and infusing a small amount of saline, removingthe catheter and squeezing to collect the fluid that returns on thenipple surface. The use of a rigid 1.2 mm ductoscope to identifyintraductal papillomas in women with nipple discharge is described inMakita et al (1991) Breast Cancer Res Treat 18: 179-188. It would beadvantageous to collect the ductal fluid from within the duct and sofacilitate duct-specific analysis.

SUMMARY OF THE INVENTION

It is an object of the invention to provide a method for preparing asample for use in diagnosis of breast cancer or pre-cancer.

It is another object of the invention to provide an isolated ductalfluid sample suitable for analyzing breast cancer and pre-cancer.

It is yet another object of the invention to provide a method foranalyzing breast markers or epithelial cells.

These and other objects of the invention are provided by one or more ofthe embodiments described below. In one embodiment a method is providedfor preparing a sample for use in the diagnosis of breast cancer orpre-cancer. A ductal fluid sample is isolated from one duct of a breastof a patient. The isolated ductal fluid is not mixed with ductal fluidfrom any other duct of the breast.

According to another embodiment of the invention an isolated ductalfluid sample is provided. The sample is collected from a breast duct ina breast. The isolated ductal fluid is not mixed with ductal fluid fromany other breast duct.

According to still another embodiment of the invention a method isprovided for analyzing breast markers or epithelial cells. The presenceor absence of a marker in an isolated ductal fluid sample is determined.The sample is collected from a breast duct in a breast. The isolatedductal fluid not mixed with ductal fluid from any other breast duct.

The present invention thus provides the art with improved samples andsampling techniques for diagnosing and prognosing breast cancer andpre-cancer.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS OF THE INVENTION

The following preferred embodiments and examples are offered by way ofillustration and not by way of limitation. The invention comprises anisolated ductal fluid sample collected from a breast duct in a breast,the fluid not mixed with ductal fluid from any other breast duct. Theisolated ductal fluid sample can be a sample from a non-dischargingbreast duct. A non-discharging duct is a breast duct that is notspontaneously discharging fluid or material, i.e., a duct which is notleaking fluid to the nipple surface. Spontaneously discharging ductsdischarge fluid of various coloration. The spontaneous discharge itselfis a warning sign usually requiring further investigation, such as,mammography, ductoscopy, and/or galactography. The present inventionprovides an isolated ductal fluid sample from a non-discharging duct,i.e., a ductal fluid and/or material sample, a portion of which wouldnot otherwise have contacted the nipple surface. However, the isolatedductal fluid sample may also be from a discharging duct, provided thesample collected is not mixed with ductal fluid from any other duct.

The isolated ductal fluid sample can be examined for the presence of amarker, the absence of a marker, or the state or quality of a particularmarker. The markers can comprise those detailed herein and relatedmarkers that indicate the status or condition of the breast. The markerstatus can be used to identify pre-cancer or cancer of the breast. Theductal fluid sample is collected from one duct of a breast of a patient.Ductal fluids may be collected from multiple ducts of a breast or fromducts in both breasts of a patient, e.g., in sequence, provided thefluid and material from each duct is kept separate for analysis from theother ducts. The ducts are also marked or otherwise identified so thatfollow-up and/or treatment of a duct that indicates the need fortreatment can be conducted. The ductal fluid sample when collected orprovided is not mixed with ductal fluid from any other duct of thebreast.

The number of epithelial cells in a ductal fluid sample may range, forexample, from a few to a hundred, to several hundred, to severalthousand, and up to tens of thousands, e.g., 20,000 to 100,000 or morecells. At least ten epithelial cells are required to designate anisolated sample as adequate for analysis of the cells. An isolatedductal fluid sample can have 10 or more cells for analysis, and possiblya single clump of cells or more than one clump. A clump comprises aplurality of cells, generally at least about 4 to about 6 cells are in aclump, and the clump can comprise more cells than 6. Samples with one ormore clumps can also include individual cells that are distinct from theclump(s). Thus, an isolated sample retrieved by infusing fluid into theduct and collecting the infused fluid mixed with the ductal fluid canprovide multiple cells and one or more cell clumps for analysis. Theadvantage of cell clumps is that the clumping provides a framework foranalysis of cell-cell interaction or a cell-to-cell relationship that inturn provides information about the status of the cells themselves. Theinvention provides the a ductal fluid sample comprising sufficientductal epithelial cells from a breast duct for an analysis of the breastin which the duct is located. Insufficient ductal epithelial cells in asample means that a cytological analysis of those cells can not beperformed, or that the accuracy of the cytological analysis iscompromised. The method of the invention and the composition providesamples from single breast ducts that can be analyzed because thesamples so isolated contain sufficient material for an adequate analysisto be made. Ductal samples can comprise markers present in the fluid inaddition to cells, i.e., molecules present in the cells collected and/orin the extracellular material retrieved from the breast duct. Anadvantage provided by the invention is that many more cells than havebeen previously collected are collectable and an accurate cytologicalanalysis can therefore be made of the sample.

Relatively undisrupted cells and clumps can be analyzed to provideinformation on the cellular status in the breast duct from which thesample was collected. Further, collection of the ductal fluid from thebreast duct provides enough cells and/or other material from the duct toprovide a useful analysis of the condition of the breast. This islargely due to the fact that collection of the ductal fluid, cells andother material by infusing saline or other biocompatible wash fluid andcollecting the wash fluid mixed with the ductal material results incollection of sufficient fluid and material for analysis. Suction may beapplied to the lumen in the duct to facilitate collection of the ductalfluid and material once the duct has been filled with wash fluid (inorder to prevent collapse of the ductal walls); reinfusion of wash fluidcan follow in order to prevent collapse of the ductal walls and providethe opportunity for a second or subsequent intraductal aspiration and/orretrieval. Squeezing and massaging the breast may also be used inconcert with infusion and collection procedures. The amount of materialthat is sufficient for analysis in a sample is at least one ductalepithelial clump up to at least 10 ductal epithelial clumps or more—eachclump having from at least 4 to 6 ductal epithelial cells. For example,the sample from a non-discharging or discharging breast duct may have atleast from 10 to 20 ductal epithelial cells, 20 to 50, 50 to 100, 100 to1000, 1000 to 10,000, 10,000 to 50,000 or 50,000 to 100,000 ductalepithelial cells. The ductal epithelial cells may be present eitherindividually or in clumps or both. Insufficient samples might includesamples with less than 10 epithelial cells. An evaluation ofinsufficiency maybe contingent on whether the cells are clumped or not.

The method of the invention is preparing a sample for use in diagnosisof breast cancer or pre-cancer comprising isolating a ductal fluidsample from one duct of a breast of a patient. The isolated ductal fluidis not mixed with ductal fluid from any other duct of the breast. Themethod can further include examining the isolated ductal fluid sample todetermine the presence or absence of a marker. The duct from which theductal fluid is isolated can be a duct that is not spontaneouslydischarging fluid. The marker for analysis can be selected from anyknown and useful markers for a breast condition, including pre-cancerand/or cancer markers, and further optionally including markers listedherein.

The isolated fluid sample can be examined to determine the presence of amarker. The presence of any marker, the absence of any marker, or thequality or state of any marker can be analyzed or examined.Particularly, the markers listed herein, and related forms or species ofthe markers listed herein are contemplated. The presence, absence orstate of more than one marker can be examined. Markers can be examinedin conjunction with ductal epithelial cell cytological analysis. Themarkers can be, for example, intracellular, nuclear, cytoplasmic,cell-surface, secreted, or extracellular markers. The markers caninclude any markers described in co-owned, co-pending, parentapplication Ser. No. 09/625,399 filed Jul. 26, 2000, application Ser.No. 09/502,404, filed on Feb. 10, 2000, and application Ser. No.09/313,463, filed on May 17, 1999, hereby incorporated by reference intheir entirety.

Examination of the ductal fluid for a marker can comprise determiningabsorption of a marker molecule by abnormal cells in the fluid. Forexample, absorption of iodide or a like molecule by cells in a ductalfluid sample can be measured. Examination of the ductal fluid for amarker can comprise analysis or examination of a quality and/or state ofnucleic acid for such characteristic changes from the normal state as,for example, a loss of heterozygozity. Examination of the ductal fluidcan comprise examining the fluid for the absence of a marker; especiallywhere the marker is present in normal ductal fluid in a predeterminedquantity in the population, and standards are set for benchmarksindicating a particular condition in the breast (i.e., pre-cancer orcancer, or their various sub-categories). Examination of the ductalfluid can comprise examining the ductal fluid for the presence orabsence of two or more markers, including examining two or more markersfor their state or quality, and including absorption of a marker, orloss of heterozygosity in the DNA of the cells retrieved from theisolated sample.

For example, the markers in this latter case can comprise DNA content,p53 gene or gene product, and G-actin or a nucleic acid encoding apolypeptide comprising at least a portion of G-actin. Thus, for example,examining the ductal fluid can comprise examining the fluid for thepresence of at least one marker and the absence of at least one marker,e.g., examining the ductafluid for the presence of an oncogene or itsgene product, and examining the ductal fluid for the absence of a tumorsuppressor molecule normally present in a given range or quantity innormal breast duct fluid or breast tissue. As an example, the ductalfluid can be examined for markers comprising such parameters as DNAcontent of the ductal epithelial cells, the absence or lowered levels ofp53 gene or its gene product, and the presence of G-actin protein,polypeptide, or portion thereof, or a nucleic acid encoding a G-actinprotein or polypeptide or portion thereof, e.g. as described in Rao etal, Cancer Epid, Biomarkers & Prevention, 1993 v. 7:1027-1033.

Preparing an isolated ductal fluid sample can comprise accessing theduct with a ductal access tool and collecting the ductal fluid samplewhile the tool remains in the duct. Having the tool remain in the ductfor fluid infusion and fluid collection also ensures that the ductalfluid and ductal material collected are collected from a single duct notmixed with fluid or material from any other duct. Wash fluid infusion isused in the cases where the duct is not spontaneously discharging fluidso that the duct is filled or partially filled, the wash fluid mixeswith ductal fluid and ductal contents, and retrieval of the mixed fluidscomprises retrieval of a sample having sufficient cells and/or othermarkers for analysis of the condition of the duct from which the sampleis taken. Previously the usefulness of ductal fluid retrieved by othermeans has been hampered by insufficient material or cells for analysisin the retrieved samples and/or not being able to identify the specificduct to which abnormal cells or other findings can be attributed. Sincemost breast cancers begin in a single, isolated, milk duct of a breast,the identification of a specific duct as abnormal (i.e., cancerous orpre-cancerous) is extremely useful, especially in concert withsufficient information from the isolated fluid sample in order to make adiagnosis. Collection of the isolated fluid sample can be facilitated byfluid infusion into the duct and collection of the wash fluid that hasbeen infused mixed with ductal fluid and other ductal contents includingductal epithelial cells and other markers. The collection through theaccessing lumen is facilitated after wash fluid infusion by a number oftechniques that can be used together or separately and which are notlimited to squeezing the breast, massaging the breast, applying negativepressure on the lumen to pull-up fluid into the lumen and/or collectionreceptacle, and using an additive in the wash fluid that delays orinhibits absorption of the fluid into the ductal walls (thereby keepingmore fluid in the duct to be retrieved). Many of these techniques andtools for practicing these techniques are described in co-owned U.S.Ser. No. 09/067,661, U.S. Ser. No. 09/301,058, PCT US99/09141, U.S. Ser.No. 09/313,463, U.S. Ser. No. 09/473,510, PCT US99/31086 hereinincorporated by reference in their entirety.

By the procedure of ductal lavage, ductal epithelial cells that line thewalls of the ductal lumen are washed out of the duct. Lavage or washfluid is infused into the duct, and the lavage fluid mixed with ductalfluid is collected. Lavage is described in copending and co-ownedapplications including Ser. No. 09/067,661, 09/301,058, PCT US99/09141,60/122,076, Ser. No. 09/313,463, 60/143,359, and U.S. Ser. No.09/473,510, all incorporated by reference in their entirety. Suction canbe applied to the tool accessing the ductal lumen in order to retrieve amaximum amount of cells and/or fluid. Lavage or wash fluid can beinfused into the duct, and collected. Suction can be applied to the toolaccessing the ductal lumen in order to retrieve a maximum amount ofcells and/or fluid. The duct can be flushed by infusing saline into theduct until resistance is met, applying pressure and/or squeezing thebreast, e.g., particularly at the base of the breast, and capturing thefluid that moves up through the duct after the pressure is applied.Flushing can continue by infusing more saline and applying morepressure.

In order to retrieve cells and ductal material sufficient for analysisof a single non-discharging breast duct and a corresponding diagnosis, anon-discharging duct can be accessed by a tool capable of infusing washfluid and also capable of collecting the ductal fluid mixed with washfluid while the tool remains in the duct as described herein. Thus,ductal fluid can be retrieved by a medical tool, e.g., a catheter or acannula, placed into the duct to infuse wash fluid to retrieve a mixtureof wash fluid and ductal fluid from the duct without removal of thetool. Thus, by the method of the invention, the tool remains indwellingwhile wash fluid is infused and wash fluid mixed with ductal fluid(comprising cells and cellular material, etc.) is collected. The fluidfrom the breast duct can contain ductal epithelial cells, includingcells of a stage considered to be pre-cancerous or cancerous asdescribed, and may also contain various molecules either connected tothe cells or separate from them that may be used as markers. Eitherpresence or absence or decrease or increase relative to a normal orbenign control amount can indicate cancer or pre-cancer.

The method is practiced by providing a ductal fluid sample from at leastone duct of a breast of the patient. Providing the ductal fluid samplecan be accomplished by obtaining the sample from the breast or byreceiving a sample that had been previously obtained. For example, alaboratory can receive a ductal fluid sample from a patient or apractitioner, and the laboratory can be directed to make an analysis ofthe sample. The isolated fluid is collected by some available techniqueincluding, for example, ductal lavage of a single duct. In general,collection of isolated ductal fluid not mixed with ductal fluid fromanother duct of the breast can be accomplished by accessing the ductwith a breast duct access tool that infuses fluid and collects ductalfluid mixed with the infused fluid, while the tool remains in the duct.Also, the collection tube can be marked and the duct can be marked sothat the analysis of the fluid is traceable to one duct which can bere-identified and re-accessed if appropriate.

The marker may be a nucleic acid or protein form of the marker. Forexample, the marker may be “X”, and either a nucleic acid sequenceencoding at least a portion of X, or a gene product at least a portionof protein or polypeptide X can be determined or measured. The markermay also be a non-nucleic acid or a non-amino acid molecule, such as,for example, a small organic molecule, a lipid, a fat, a biologicallyformed organic acid or base, a carbohydrate or other sugar type moleculea polymer type molecule or a portion of such molecule, a moiety thatcharacterizes a marker, for example a side chain on a marker, etc.

Thus, for example, the method of providing an isolated ductal fluidsample, and examining the sample for one or more markers can compriseexamining the sample for the presence, absence, or relative level of anyone or more of the following markers:

-   -   1. lysophosphatidic acid (LPA) or a lysophospholipid, or a        receptor of lysophosphatidic acid, e.g., as described in Goetzl        et al, Cancer Res Sep. 15, 1999 v. 59: 4732-7, Xu et al, Biochem        J 1995 v. 309: 933-40, and Contos et al, Mol Pharmacol 2000 v.        58: 1188-1196;    -   2. palladin, a portion of palladin, or a nucleic acid encoding a        polypeptide comprising at least a portion of paladin, e.g., as        described in Reuters Health News Aug. 7, 2000, and Parast and        Otey, J Cell Biol Aug. 7, 2000 v. 150:643-56;    -   3. Lg, a portion of Lg, or a nucleic acid encoding a polypeptide        comprising at least a portion of Lg, e.g., as described in        Ranganathan et al, J Steroid Biochem Mol Biol 1999 v. 70: 151-8;    -   4. E2F1, a portion of E2F1, or a nucleic acid encoding a        polypeptide comprising at least a portion of E2F1, e.g., as        described in Klein-Szanto et al Cancer Epid Biomarkers &        Prevention 2000 v. 9: 395-401;    -   5. T1A12/mac 25, a portion of T1A12/mac 25, or a nucleic acid        encoding a polypeptide comprising at least a portion of        T1A12/mac 25, e.g., as described in Burger et al Oncogene        1998 v. 16:2459-67;    -   6. MAGUK/ZO-1, a portion of MAGUK/ZO-1, a nucleic acid encoding        a polypeptide comprising at least a portion of MAGUK/ZO-1, e.g.,        as described in Hoover et al, Am J Pathol 1998 v. 153: 1767-73;    -   7. Repressor of estrogen receptor activity (REA), a portion of        REA, a nucleic acid encoding a polypeptide comprising at least a        portion of REA, e.g., as described in Simon et al, Cancer Res        2000 v. 60:2796-9;    -   8. prothymosin alpha (PTA), a portion of PTA, a nucleic acid        encoding a polypeptide comprising at least a portion of PTA,        e.g., as described in Domineguez et al, Br J of Cancer 2000 v.        82:584-590; and Magdalena et al, Br J Cancer 2000 v. 82:584-590;    -   9. TNF-related apoptosis-inducing ligand (TRAIL), a nucleic acid        encoding a polypeptide comprising at least a portion of TRAIL,        e.g., as described in Griffith et al, J Immunol 2000 v.        165:2886-94; and Herrnring et al Histochem Cell Biol 2000 v.        113:189-94;    -   10. BU101 protein, a nucleic acid encoding a polypeptide        comprising at least a portion of BU101, e.g., as described in WO        98/07857;    -   11. c-raf kinase, a portion of c-raf kinase, a nucleic acid        encoding a polypeptide comprising at least a portion of c-raf        kinase, e.g., as described in El-Ashry et al, Oncogene 1997 v.        15: 423-35, and Callans et al, Ann Surg. Oncol 1995 v. 2: 38-42;    -   12. CD66a, a portion of CD66a, a nucleic acid encoding a        polypeptide comprising at least a portion of CD66a, e.g., as        described in Huang et al Anticancer Res. 1998 v. 18: 3203-12;    -   13. KL-1, a portion of KL-1, a nucleic acid encoding a        polypeptide comprising at least a portion of KL-1, e.g., as        described in Okumura et al, Jpn J Clin Oncol 1998 v. 28: 480-5;    -   14. cell adhesion molecule 5.2 (CAM 5.2), a portion of CAM 5.2,        a nucleic acid encoding a polypeptide comprising at least a        portion of CAM 5.2, e.g., as described in Okumura et al, Jpn J        Clin Oncol 1998 v. 28: 480-5;    -   15. leptin, a portion of leptin, a nucleic acid encoding a        polypeptide comprising at least a portion of leptin, e.g., as        described in Tessitore et al, Int J Mol Med 2000 v. 5: 421-6;    -   16. Bcl-2 gene product, at least a portion of Bcl-2 gene product        or polypeptide, a nucleic acid encoding a polypeptide encoding        at least a portion of Bcl-2 gene product, e.g., as described in        Krajewski et al Endocr Relat Cancer 1999 v. 6: 29-40, and        Castiglione et al Anticancer Res. 1999 v. 19:4555-63,        Simony-Lafontaine et al, 2000-v. 82: 1958-66;    -   17. nuclear matrix 23(nm23), a portion of nm23, a nucleic acid        encoding a polypeptide comprising at least a portion of nm23,        e.g., as described in Vazquez-Ramirez et al, Pathol Res Pract        2000 v. 196: 553-9, Cipollini et al, Cancer Genet Cytogenet        2000 v. 121:181-5;    -   18. an apotosis-related protein, a portion of said protein, a        nucleic acid encoding a polypeptide comprising at least a        portion of the apoptosis-related protein, e.g., as described in        Dowsett et al, Endocr Relat Cancer 1999 v. 6: 25-8;    -   19. lipocalin NGAL, a portion of lipocalin NGAL, a nucleic acid        encoding a polypeptide comprising at least a portion of        lipocalin NGAL, e.g., as described in Stoesz et al, Int J Cancer        1998 v. 79: 565-72;    -   20. thymosin beta-15, a portion of thymosin beta-15, a nucleic        acid encoding a polypeptide comprising at least a portion of        thymosin bet-15, e.g., as described in U.S. Pat. No. 5,663,071        and WO 97/48805;    -   21. tumor amplified kinase STK15 (also BTAK and aurora2), at        least a portion of STK15, a nucleic acid encoding at least a        portion of STK15; e.g., as described in Zbou et al, Nat Genet        1998 v. 20:189-93;    -   22. complement regulatory protein CD 46, a portion of CD46, a        nucleic acid encoding at least a portion of CD46; as described        e.g., in Thorsteinsson et al APMIS 1998 v. 106:869-78;    -   23. complement regulatory protein CD 59, a portion of CD 59, a        nucleic acid encoding at least a portion of CD59; as described        e.g., in Thorsteinsson et al APMIS 1998 v. 106:869-78;    -   24. a nucleic acid encoding a portion of an FHIT gene, e.g., as        described in Huiping et al, Eur J Cancer 2000 v. 36: 1552-7,        Gatalica et al, Cancer 2000 v. 88: 1378-83, Ahmadian et al,        Cancer Res 1997 v. 57:3664-8, and Campiglio et al, Cancer Res        1999 v. 59:3866-9;    -   25. loss of heterozygosity (LOH), e.g., as described in        Linginger et al, Mod Pathol 1998 v. 11:1151-9; and Larson et al,        Am J Pathol 1998 v. 152:1591-8;    -   26. LOH at an FRA3B site, e.g., as described in Ahmadian et al,        Cancer Res 1997 v. 57:3664-8;    -   27. MRP-1/CD9, a portion of MRP-1/CD9, a nucleic acid encoding        at least a portion of MRP-1/CD9, e.g., as described in van den        Heuvel-Eibrink et al, Int J Clin Pharmacol Ther 2000 v.        38:94-110, Huang et al Am J Pathol 1998 v. 153: 973-83, Miyake        et al Cancer Res 1996 v. 56:1244-9, and Miyake et al Cancer Res.        1995 v. 55: 4127-31;    -   28. KAI1/CD82, a portion of KAI1/CD82, a nucleic acid encoding        at least a portion of KAI1/CD82, e.g., as described in Huang et        al Am J Pathol 1998 v. 153: 973-83;    -   29. TM S-1, a portion of TMS-1, a nucleic acid encoding a        polypeptide comprising at least a portion of TMS-1, for example        as described in McConnell and Vertino, Cancer Res. Nov. 15,        2000;60(22):6243-7; Conway et al, Cancer Res Nov. 15,        2000;60(22):6236-42; and Grossman et al, J Exp Biol 2000 v. 203:        447-57;    -   30. at least a portion of breast cancer associated gene (BRCA),        e.g., as described in Seances et al, Soc. Biol Fil 1998 v.        192:35-40, and Deng and Brodie, Bioessays 2000 v. 22: 728-37;    -   31. absorption of a marker (e.g., iodide), e.g., as described in        De La Vieja et al, Physiol Rev 2000 v. 80: 1083-105, Tazebay et        al, Nat Med 2000 v. 6:871-8;    -   32. Fibroblast growth factor (FGF) protein, a portion of an FGF        protein or polypeptide, a nucleic acid encoding at least a        portion of an FGF protein or polypeptide, e.g. as described in        Fernig et al, Cancer Treat Res 1991 v. 53: 47-78, and De        Benedetti and Harris, Int J Biochem Cell Biol 1999 v. 31: 59-72;    -   33. Vascular endothelial growth factor (VEGF) protein, a portion        of a VEGF protein or polypeptide, a nucleic acid encoding at        least a portion of a VEGF protein or polypeptide e.g., as        described in Gasparini, Oncologist 2000; 5 suppl 1:37-44;    -   34. Insulin-like growth factor-1 (IGF-1 protein, a portion of an        IGF-1 protein or polypeptide, a nucleic acid encoding at least a        portion of an IGF-1 protein or polypeptide e.g., as described in        Pollack, Eur J Cancer 2000 v. 36:1224-8;    -   35. Maspin protein, a portion of a maspin protein or        polypeptide, a nucleic acid encoding at least a portion of a        maspin protein or polypeptide, e.g., as described in Sager et        al, Adv Exp Med Biol 1997 v. 425: 77-88.    -   36. CDw60 protein, a portion of CDw60 protein or polypeptide, a        nucleic acid encoding at least a portion of a CDw60 protein or        polypeptide, e.g., as described in Gocht et al, Histochem J July        2000;32(7):447-56.    -   37. Mammary expressed enzymes (e.g., cytochrome P450s,        catechol-O-methyltransferase, epoxide hydrolase, peroxidases,        glutathione S-transferases, N-acetyltransferases, and        sulfotransferases) a nucleic acid encoding at least a portion of        a mammary expressed enzyme, e.g., as described in Williams and        Phillips, Cancer Res Sep. 1, 2000:60(17):4667-77.    -   38. Mammastatin protein or polypeptide (47 kD and/or 65 kD), a        nucleic acid encoding at least a portion of a mammastatin        protein or polypeptide, e.g., as described in Ervin et al,        Science 1989; 244(4912); 1585-7.    -   39. Kallikrein 6 (zyme/protease M/neurosin) protein or        polypeptide (hK6), a nucleic acid encoding at least a portion of        an hK6 protein or polypeptide, e.g., as described in Diamandis        et al, Clin Biochem October 2000; 33(7):579-583; Diamandis et        al, Clin Biochem July 2000;33(5):369-75; Yousef et al, Genomics        Nov. 1, 2000;69(3):331-41.

As discussed, the cells collected can comprise ductal epithelial cellsand the ductal fluid collected can comprise molecular and cellularmaterial. The collected cells and fluid and fluid components can beanalyzed, e.g., as described or suggested herein. Fluid collected fromthe milk ducts, can include constituents of biological fluids, e.g.,those typically found in breast duct fluid, e.g., water, cells, cellularmarkers, molecular markers, nucleic acids, proteins, cellular debris,salts, particles or organic molecules. These constituents can beanalyzed by any appropriate method depending on the marker and thediagnostic purpose. In addition, any of the cells of the duct can beanalyzed for morphological abnormalities in cell components, including,e.g., morphological abnormalities of the nucleus, cytoplasm, Golgiapparatus or other parts of a cell. Cell morphology can serve toestablish whether the ductal epithelial cells are normal (i.e., notpre-cancerous or cancerous or having another noncancerous abnormality),pre-cancerous (i e., comprising hyperplasia, atypical ductal hyperplasia(ADH) or low grade ductal carcinoma in situ (LG-DCIS)) or cancerous(i.e., comprising high grade ductal carcinoma in situ (HG-DCIS), orinvasive carcinoma). Analysis of cell contents may serve to establishsimilar staging as established by morphology, capturing generally aprogression of a pre-cancerous or cancerous condition in the cells.

Once the ductal fluid sample is retrieved from the breast it is examinedfor the presence of a marker such as, for example a protein, apolypeptide, a peptide, a nucleic acid, a polynucleotide, an mRNA, asmall organic-molecule, a lipid, a fat, a glycoprotein, a glycopeptide,a carbohydrate, an oligosaccharide, and a chromosomal abnormality, awhole cell having a marker molecule, a particle, a secreted molecule, anintracellular molecule, and a complex of a plurality of molecules asdescribed above. In addition, the marker may be capable ofdistinguishing between any two cytological categories consisting ofnormal, abnormal, hyperplasia, atypia, ductal carcinoma, ductalcarcinoma in situ (DCIS), ductal carcinoma in situ—low grade (DCIS-LG),ductal carcinoma in situ—high grade (DCIS-HG), invasive carcinoma,atypical mild changes, atypical marked changes, atypical ductalhyperplasia (ADH), insufficient cellular material for diagnosis, andsufficient cellular material for diagnosis. These categories classifythe epithelial cells cytologically, and these classifications mayindicate either cancer or its precursors, or absence of cancer indicia.

Analysis of cell contents may serve to establish similar staging asestablished by morphology, capturing generally a progression of apre-cancerous or cancerous condition in the cells. Thus the ductalepithelial cells may be analyzed for other markers, e.g.,, proteinmarkers, nucleic acid markers, particles, complexes, or biochemical ormolecular markers in the cells or on the cell surfaces or secreted bythe cell or for any marker providing evidence of neoplasia. The ductalepithelial cell can be derived from any part of the breast milk duct,including, e.g.,, the ductal lumen and/or the terminal ductal lobularunit (TDLU). Cells derived from the TDLU may also have similar stages asfound in other lumenal ductal epithelial cells not from the TDLUincluding, e.g.,, hyperplasia, atypia, in situ carcinoma, and invasivecarcinoma.

Once the wash fluid has been infused in the duct and the wash fluid andductal fluid is collected from a breast duct, the cellular material canbe separated and can be examined. The cellular material can include,e.g., substances selected from the group consisting of whole cells,cellular debris, proteins, nucleic acids, polypeptides, glycoproteins,lipids, fats, glycoproteins, small organic molecules, metabolites, andmacromolecules. These materials may be found in the cell, on the cellsurface or as material secreted from the cell and found in fluid outsidethe cell. These materials may be synthesized by a cell, or may beotherwise present in the fluid from the duct, e.g., as by-products ordegradation products of molecules in the body. Cytology, or any othersuitable method for analyzing the condition of the cells can be used toexamine whole cells Other markers present in the cellular material,ductal fluid, or other material obtained from the breast duct can beanalyzed as is appropriate for the marker being sought, including e.g.,binding assays, immunohistochemistry, or using other analyticaltechniques for distinguishing and identifying biological moleculesobtained from biological material. Examining the ductal fluid sample canalso be performed to determine the presence of a marker comprising, forexample, a protein, a polypeptide, a peptide, a nucleic acid, apolynucleotide, an mRNA, a small organic molecule, a lipid, a fat, aglycoprotein, a glycopeptide, a carbohydrate, an oligosaccharide, achromosomal abnormality, a whole cell having a marker molecule, aparticle, a secreted molecule, an intracellular molecule, or a complexof a plurality of molecules. Detection and analysis of theseclassifications of markers can be accomplished, for example, usingstandard assays for determining the presence of a particular marker ormarker classification and/or for example as described in Sambrook etal., Molecular Cloning: A Laboratory Manual, 2^(nd) Ed. (Cold SpringHarbor Press, Cold Spring Harbor, N.Y. 1989).

Examining the ductal fluid sample can comprise determining the presenceof a marker comprising RNA, DNA, protein, polypeptide, or peptide formof a marker such as lysophosphatidic acid, a lysophospholipid, paladin,a portion of palladin, a nucleic acid encoding a polypeptide comprisingat least a portion of paladin, Lg, a portion of Lg, a nucleic acidencoding a polypeptide comprising at least a portion of Lg, E2F1, aportion of E2F1, a nucleic acid encoding a polypeptide comprising atleast a portion of E2F1, T1A12/mac 25, a portion of T1A12/mac 25, anucleic acid encoding a polypeptide comprising at least a portion ofT1A12/mac 25, MAGUK/ZO-1, a portion of MAGUK/ZO-1, a nucleic acidencoding a polypeptide comprising at least a portion of MAGUK/ZO-1,repressor of estrogen receptor activity (REA), a portion of REA, anucleic acid encoding a polypeptide comprising at least a portion ofREA, prothymosin alpha (PTA), a portion of PTA, a nucleic acid encodinga polypeptide comprising at least a portion of PTA, c-raf kinase, aportion of c-raf kinase, a nucleic acid encoding a polypeptidecomprising at least a portion of c-raf kinase, CD66a, a portion ofCD66a, a nucleic acid encoding a polypeptide comprising at least aportion of CD66a, KL-1, a portion of KL-1, a nucleic acid encoding apolypeptide comprising at least a portion of KL-1, cell adhesionmolecule 5.2(CAM 5.2), a portion of CAM 5.2, a nucleic acid encoding apolypeptide comprising at least a portion of CAM 5.2, leptin, a portionof leptin, a nucleic acid encoding a polypeptide comprising at least aportion of leptin, Bcl-2 gene product, at least a portion of Bcl-2 geneproduct or polypeptide, a nucleic acid encoding a polypeptide encodingat least a portion of Bcl-2 gene product, nuclear matrix 23(nm23), aportion of nm23, a nucleic acid encoding a polypeptide comprising atleast a portion of nm23, an apotosis-related protein, a portion of saidprotein, a nucleic acid encoding a polypeptide comprising at least aportion of the apoptosis-related protein, comprises lipocalin NGAL, aportion of lipocalin NGAL, a nucleic acid encoding a polypeptidecomprising at least a portion of lipocalin NGAL, a nucleic acid encodinga portion of an FHIT gene, loss of heterozygosity at an FRA3B site,MRP-1/CD9, a portion of MRP-1/CD9, a nucleic acid encoding at least aportion of MRP-1/CD9, KAI1/CD82, a portion of KAI1/CD82, a nucleic acidencoding at least a portion of KAI1/CD82, at least a portion of breastcancer associated gene, TMS-1, a portion of TMS-1, a nucleic acidencoding a polypeptide comprising at least a portion of TMS-1; at leasta portion of breast cancer associated gene (BRCA); absorption of amarker (e.g., iodide) fibroblast growth factor (FGF) protein, a portionof an FGF protein or polypeptide, a nucleic acid encoding at least aportion of an FGF protein or polypeptide, vascular endothelial growthfactor (VEGF) protein, a portion of a VEGF protein or polypeptide, anucleic acid encoding at least a portion of a VEGF protein orpolypeptide, insulin-like growth factor-1 (IGF-1 protein, a portion ofan IGF-1 protein or polypeptide, a nucleic acid encoding at least aportion of an IGF-1 protein or polypeptide; maspin protein, a portion ofa maspin protein or polypeptide, a nucleic acid encoding at least aportion of a maspin protein or polypeptide, CDw60 protein, a portion ofCDw60 protein or polypeptide, a nucleic acid encoding at least a portionof a CDw60 protein or polypeptide, mammary expressed enzymes. (e.g.,cytochrome P450s, catechol-O-methyltransferase, epoxide hydrolase,peroxidases, glutathione S-transferases, N-acetyltransferases, andsulfotransferases) a nucleic acid encoding at least a portion of amammary expressed enzyme, mammastatin protein or polypeptide (47 kDand/or 65 kD), a nucleic acid encoding at least a portion of mammastatinprotein or polypeptide; kallikrein 6 (zyme/protease M/neurosin) proteinor polypeptide (hK6), and a nucleic encoding at least a portion of anhK6 protein or polypeptide.

A level of the marker can be a presence relative to a normal control oran absence relative to a normal control of a given marker. Increased ordecreased amounts relative to such normal controls can also bedetermined. The normal control can be determined relative to theparticular patient, or relative to a patient population. In addition,the quality of the marker can be assessed. A quality of a marker can besuch changes as DNA mutation, or a quantity of mutations, adeterioration of chromosomal quality or quantity, degradation of aprotein, or a change in quantity of a nucleic acid or chromosome. Aquality can be an erosion of a molecule, particle, molecule or organellewith respect to a normal quality. A tumor suppressor, e.g., mammastatinmay be used as a marker where a reduction in the marker identifies acancerous or pre-cancerous condition in the breast.

Chromosomal abnormalities in ductal epithelial cells can also provideinformation and act as a marker to identify cancer or pre-cancer asdescribed in Mark et al (1999) Cancer Genet Cytogenet 108:26-31; Lundlinand Mertens (1998) Breast Cancer Res Treat 51:1-15; Newsham (1998) Am JPathol 153:5-9; Larson et al (1998) Am J Pathol 152:1591-8; Adelaide etal (1998) Genes Chromosomes Cancer 22:186-99; Fejzo et al (1998) GeneChromosome Cancer 22:105-113; Dietrich et al (1998) Hum Pathol 12:1379-82; Cavalli et al (1997) Hereditas 126:261-8; Adeyinka et al (1997)Cancer Genet Cytogenet 97:119-21; Afify and Mark (1997) Cancer GenetCytogenet 97:101-5; Brenner and Aldaz (1997) Prog Clin Biol Res 396:63-82; Mark et al (1997) Ann Clin Lab Sci 27:47-56; and Fabian et al1993 J.; Cellular Biochemistry 17G:153-16.

Standard assay procedures for identifying the markers can be used,including antibodies or other binding partners, labels, stains, patternanalysis (for cells and cell components), and in general any otherchemical or visual identification techniques.

The different categories of markers are tested differently depending onthe category and possibly also on the location of the marker in the cell(for example, a cell surface protein might be detected differently thana cytoplasmic or nuclear protein). Typically, assays comprising one ormore of binding, coloration, precipitation, affinity column selection,in-situ binding, solution phase binding, nucleic acid probe labeling,protein probe labeling, polypeptide probe labeling, peptide probelabeling, and/or a combination or variation of these processes can beused. Standard procedures for conducting such assays generally (e.g.,ELISA, RNA or DNA probe hybridization, and other binding or otherdetection assays) are described in Sambrook et al., Molecular Cloning: ALaboratory Manual, 2^(nd) Ed. (Cold Spring Harbor Press, Cold SpringHarbor, N.Y. 1989). Standard assay procedures for identifying themarkers can be used, including antibodies or other binding partners,labels, stains, pattern analysis (for cells and cell components), and ingeneral any other chemical or visual identification techniques.

In general, markers can be categorized nonexclusively, and often inoverlapping categories, for example, protein expression, mRNAexpression, post-translational change in a protein and/or DNA change ina gene may all be used in concert or separately for the same or aplurality of markers to make a diagnosis.

Cytology, or any other suitable method for analyzing the condition ofthe cells can be used to examine whole cells. Markers present in thecellular material, ductal fluid generally, or other material obtainedfrom the breast duct can be analyzed as is appropriate for the markerbeing sought, including, e.g., binding assays, immunohistochemistry, orusing other analytical techniques for distinguishing and identifyingbiological molecules obtained from biological material

Once the ductal fluid is analyzed for one or more markers, the fluid mayalso be analyzed cytologically to determine the cytological status ofthe ductal epithelial cells and other cells. Cytological assays that canbe performed on the cells retrieved from a duct or from nipple aspiratecan include e.g., assays described in King et al, J. Nat'l Cancer Inst(1983) 71:1115-21, Wrensch et al. (1992) Am. J. Epidem. 135: 130-141,Papanicolaou et al, (1958) Cancer, 11:377-409 and Goodson W H & King EB, Chapter 4: Discharges and Secretions of the Nipple, THE BREAST:COMPREHENSIVE MANAGEMENT OF BENIGN AND MALIGNANT DISEASES (1998) 2^(nd)Ed. vol 2, Bland & Kirby eds. W. B. Saunders Co, Philadelphia, Pa. pp.51-74. For example, as described in Goodson and King (page 60) atypicalhyperplasia presents as having cellular abnormalities, increasedcoarseness of the chromatin, and tendency for more single cells as wellas groups of cells. With regard to carcinoma in situ, Papanicolaou et aldescribed cellular abnormalities, e.g., nuclear abnormalities diagnosedby cytology of fluid from nipple secretions containing ductal cells. Thecytological examination of abnormal cells can also be conducted asdescribed in Sartorius et al (1977) J. Natl Cancer Inst 59: 1073-1080,and King et al, (1983) JNCI 71(6) 1115-1121. Atypia and carcinoma insitu are widely characterized pathologically, as described in Page etal, (1998) Mod Pathol 11(2): 120-8. The ductal fluid can be analyzed bycytological techniques by placing some of the fluid on a slide with astandard cytological stain and observing under a light microscope. Thecells can be studied for atypical growth patterns in individual cellsand clusters of cells using published methods, including Mouriquand J,(1993) S Karger Pub, “Diagnosis of Non-Palpable Breast Lesions:Ultrasonographically Controlled Fine-Needle Aspiration: Diagnostic andPrognostic Implications of Cytology” (ISBN 3805557477); Kline T S and IK, Pub Igaku-Shoin Medical “Breast: Guides to Clinical AspirationBiopsy” (LSBN 0896401596; Masood, American Society of ClinicalPathology: November 199S, “Cytopathology of the Breast” ISBN 0891893806;and Feldman P S, American Society of Clinical Pathology, November 1984,“Fine Needle Aspiration Cytology and Its Clinical Applications: Breastand Lung” ISBN 0891891846.

Other references that discuss cytological analysis and which giveguidance to an analysis of ductal epithelial cells derived from ductalfluid include Silverman et al, (Can FNA biopsy separate atypicalhyperplasia, carcinoma in situ, and invasive carcinoma of the breast?Cytomorphologic criteria and limitations in diagnosis, DiagnosticCytopathology) 9(6): 713-28, 1993; Masood et al, (Immunohistochemicaldifferentiation of atypical hyperplasia vs. carcinoma in situ of thebreast) Cancer Detection & Prevention. 16(4): 225-35, 1992; Masood etal, (Cytologic differentiation between proliferative andnonproliferative breast disease in mammographically guided fine-needleaspirates) Diagnostic Cytopathology. 7 (6): 581-90, 1991; Masood S.,(Occult breast lesions and aspiration biopsy: a new challenge)Diagnostic Cytopathology. 9(6): 613-4, 1993; Masood S., (Prognosticfactors in breast cancer: use of cytologic preparations) DiagnosticCytopathology. 13(5): 388-95, 1995, Novak and Masood, (Nuclear groovesin fine-needle aspiration biopsies of breast lesions: do they have anysignificance?) Diagnostic Cytopathology. 18(5): 333-7, 1998; Sidawy etal, (Interobserver variability in the classification of proliferativebreast lesions by fine-needle aspiration: results of the PapanicolaouSociety of Cytopathology Study) Diagnostic Cytopathology. 18(2): 150-65,1998; Masood et al, (Automation in cytology: a survey conducted by theNew Technology Task Force, Papanicolaou Society of Cytopathology)Diagnostic Cytopathology. 18(1): 47-55, 1998; and Frykberg and MasoodCopeland EM 3d. Bland K I., (Ductal carcinoma in situ of the breast)Surgery, Gynecology & Obstetrics 177(4): 425-40, 1993.

The invention also provides systems for preparing a sample for use indiagnosis of breast cancer or pre-cancer, the system comprising a toolto retrieve ductal fluid from a breast duct and instructions for use toisolate a ductal fluid sample from a duct, particularly anon-spontaneously discharging breast duct in order to determine thepresence of one or more markers. Materials and instructions may also beincluded in the system to determine the presence or absence of a markerin the isolated ductal fluid. Materials may be included to make acytodiagnosis of collected ductal epithelial cells. A cytologicalreading of the ductal epithelial cells collected with the infused washfluid is one type of marker which can be used for diagnosing a conditionin a breast duct. Instructions in the kit or system can include guidancefor interpreting cytological data and/or other marker data in order tomake a diagnosis. The systems or kits may include a ductal access tool,for example in order to retrieve the ductal fluid, e.g., especiallywhere it is preferred that the ductal fluid be identified as coming froma specific duct (so that the duct can be accessed later for treatmentand/or further monitoring). Methods for identifying thenon-spontaneously discharging duct may also be included in the system orkit. The instructions in the systems or kits can include directionsaccording to the methods of identifying breast cancer or pre-cancerdescribed herein, and possibly including any marker or markers or markerclassification group or groups that could be useful and/or are describedherein. The system or kit can include assay reagents for detecting themarker or markers. The system or kit may comprise a panel of reagentsfor detecting a plurality of markers either simultaneously orsequentially, or some other practical combination of testing modalities.The system or kit can also include indexes and parameters for making adiagnosis, depending on the marker or markers. The system or kit caninclude a container for the contents of the system or kit.

EXAMPLES

Retrieval of Ductal Fluid and Analysis of Markers in the Fluid

A patient is prepared for a ductal access procedure. Using a ductalaccess tool, a duct on each breast is infused with sufficient washfluid, and the wash fluid mixed with ductal fluid is collectedseparately from each accessed duct. The fluid in each duct that isaccessed is analyzed for the presence, absence or relative levels (ascompared to a predetermined normal level) of one or more of thefollowing markers using standard techniques: lysophosphatidic acid, alysophospholipid, paladin, a portion of palladin, a nucleic acidencoding a polypeptide comprising at least a portion of paladin, Lg, aportion of Lg, a nucleic acid encoding a polypeptide comprising at leasta portion of Lg, E2F1, a portion of E2F1, a nucleic acid encoding apolypeptide comprising at least a portion of E2F1, T1A12/mac 25, aportion of T1A12/mac 25, a nucleic acid encoding a polypeptidecomprising at least a portion of T1A12/mac 25, MAGUK/ZO-1, a portion ofMAGUK/ZO-1, a nucleic acid encoding a polypeptide comprising at least aportion of MAGUK/ZO-1, repressor of estrogen receptor activity (REA), aportion of REA, a nucleic acid encoding a polypeptide comprising atleast a portion of REA, prothymosin alpha (PTA), a portion of PTA, anucleic acid encoding a polypeptide comprising at least a portion ofPTA, TNF-related apoptosis-inducing ligand (TRAIL), a nucleic acidencoding a polypeptide comprising at least a portion of TRAIL, BU101protein, a nucleic acid encoding a polypeptide comprising at least aportion of BU101, c-raf kinase, a portion of c-raf kinase, a nucleicacid encoding a polypeptide comprising at least a portion of c-rafkinase, CD66a, a portion of CD66a, a nucleic acid encoding a polypeptidecomprising at least a portion of CD66a, KL-1, a portion of KL-1, anucleic acid encoding a polypeptide comprising at least a portion ofKL-1, cell adhesion molecule 5.2 (CAM 5.2), a portion of CAM 5.2, anucleic acid encoding a polypeptide comprising at least a portion of CAM5.2, leptin, a portion of leptin, a nucleic acid encoding a polypeptidecomprising at least a portion of leptin, Bcl-2 gene product, at least aportion of Bcl-2 gene product or polypeptide, a nucleic acid encoding apolypeptide encoding at least a portion of Bcl-2 gene product, nuclearmatrix 23(nm23), a portion of nm23, a nucleic acid encoding apolypeptide comprising at least a portion of nm23, an apotosis-relatedprotein, a portion of said protein, a nucleic acid encoding apolypeptide comprising at least a portion of the apoptosis-relatedprotein, comprises lipocalin NGAL, a portion of lipocalin NGAL, anucleic acid encoding a polypeptide comprising at least a portion oflipocalin NGAL, thymosin beta-15, a portion of thymosin beta-15, anucleic acid encoding a polypeptide comprising at least a portion ofthymosin beta-15, a nucleic acid encoding a portion of an FHIT gene,loss of heterozygosity at an FRA3B site, MRP-1/CD9, a portion ofMRP-1/CD9, a nucleic acid encoding at least a portion of MRP-1/CD9,KAI1/CD82, a portion of KAI1/CD82, a nucleic acid encoding at least aportion of KAI1/CD82, at least a portion of breast cancer associatedgene, TMS-1, a portion of TMS-1, a nucleic acid encoding a polypeptidecomprising at least a portion of TMS-1; at least a portion of breastcancer associated gene (BRCA); absorption of a marker (e.g., iodide)fibroblast growth factor (FGF) protein, a portion of an FGF protein orpolypeptide, a nucleic acid encoding at least a portion of an FGFprotein or polypeptide, vascular endothelial growth factor (VEGF)protein, a portion of a VEGF protein or polypeptide, a nucleic acidencoding at least a portion of a VEGF protein or polypeptide,insulin-like growth factor-1 (IGF-1 protein, a portion of an IGF-1protein or polypeptide, a nucleic acid encoding at least a portion of anIGF-1 protein or polypeptide; maspin protein, a portion of a maspinprotein or polypeptide, a nucleic acid encoding at least a portion of amaspin protein or polypeptide, CDw60 protein, a portion of CDw60 proteinor polypeptide, a nucleic acid encoding at least a portion of a CDw60protein or polypeptide, mammary expressed enzymes (e.g., cytochromeP450s, catechol-O-methyltransferase, epoxide hydrolase, peroxidases,glutathione S-transferases, N-acetyltransferases, and sulfotransferases)a nucleic acid encoding at least a portion of a mammary expressedenzyme, mammastatin protein or polypeptide (e.g., 47 kD and/or 65 kD), anucleic acid encoding a mammastatin protein or polypeptide; kallikrein 6(zyme/protease M/neurosin) protein or polypeptide (hK6), and a nucleicacid encoding at least a portion of an hK6 protein or polypeptide.Cytodiagnosis of a sample of the collected ductal epithelial cells, asindividual cells and as cells in clumps is also made to support anyother marker data from the collected fluid and material.

All publications and patent applications cited in this specification areherein incorporated by reference as if each individual publication orpatent application were specifically and individually indicated to beincorporated by reference. Although the foregoing invention has beendescribed in some detail by way of illustration and example for purposesof clarity of understanding, it will be readily apparent to those ofordinary skill in the art in light of the teachings of this inventionthat certain changes and modifications may be made thereto withoutdeparting from the spirit or scope of the appended claims.

1-30. (canceled)
 31. A method to aid in diagnosing breast cancer orpre-cancer comprising: sequentially placing a ductal access toolcomprising a single lumen in at least two breast ducts of a singlebreast of a patient, wherein the single lumen has an inner diameterlarge enough to retrieve clusters of greater than 10 cells; infusing afluid into the duct through the single lumen; and retrieving ductalfluid samples from each of the sequentially accessed ducts through thesingle lumen, wherein the ductal fluid samples comprise ductalepithelial cells and are not mixed with ductal fluid samples from anyother ducts of the breast.
 32. A method as in claim 1, furthercomprising: examining the ductal fluid samples to determine the presenceor absence of a marker.
 33. A method as in claim 1, wherein the ductsfrom which the ductal fluid samples are retrieved are not spontaneouslydischarging ductal fluid.
 34. A method as in claim 32, wherein themarker is selected from the group consisting of: lysophosphatidic acid,a lysophospholipid, palladin, a portion of palladin, a nucleic acidencoding a polypeptide comprising at least a portion of palladin Lg, aportion of Lg, a nucleic acid encoding a polypeptide comprising at leasta portion of Lg, E2F1, a portion of E2F1, a nucleic acid encoding apolypeptide comprising at least a portion of E2F1, T1A12/mac 25, aportion of T1A12/mac 25, a nucleic acid encoding a polypeptidecomprising at least a portion of T1A12/mac 25, MAGUK/ZO-1, a portion ofMAGUK/ZO-1, a nucleic acid encoding a polypeptide comprising at least aportion of MAGUK/ZO-1, repressor of estrogen receptor activity (REA), aportion of REA, a nucleic acid encoding a polypeptide comprising atleast a portion of REA, prothymosin alpha (PTA), a portion of PTA, anucleic acid encoding a polypeptide comprising at least a portion ofPTA, c-raf kinase, a portion of c-raf kinase, a nucleic acid encoding apolypeptide comprising at least a portion of c-raf kinase, CD66a, aportion of CD66a, a nucleic acid encoding a polypeptide comprising atleast a portion of CD66a, KL-1, a portion of KL-1, a nucleic acidencoding a polypeptide comprising at least a portion of KL-1, celladhesion molecule 5.2 (CAM 5.2), a portion of CAM 5.2, a nucleic acidencoding a polypeptide comprising at least a portion of CAM 5.2, leptin,a portion of leptin, a nucleic acid encoding a polypeptide comprising atleast a portion of leptin, Bcl-2 gene product, a portion of Bcl-2 geneproduct, a nucleic acid encoding a polypeptide comprising at least aportion of Bcl-2 gene product, nuclear matrix 23 (nm23), a portion ofnm23, a nucleic acid encoding a polypeptide comprising at least aportion of nm23, an apoptosis-related protein, a portion of saidprotein, a nucleic acid encoding a polypeptide comprising at least aportion of the apoptosis-related protein, lipocalin NGAL, a portion oflipocalin NGAL, a nucleic acid encoding a polypeptide comprising atleast a portion of lipocalin NGAL, complement regulatory protein CD 46,a portion of CD 46, a nucleic acid encoding a polypeptide comprising atleast a portion of CD 46, complement regulatory protein CD 59, a portionof CD 59, a nucleic acid encoding a polypeptide comprising at least aportion of CD 59, a nucleic acid encoding a portion of an FHIT gene,loss of heterozygosity at an FRA3B site, MRP-1/CD9, a portion ofMRP-1/CD9, a nucleic acid encoding a polypeptide comprising at least aportion of MRP-1/CD9, KAI1/CD82, a portion of KAI1/CD82, a nucleic acidencoding a polypeptide comprising at least a portion of KAI1/CD82, aFibroblast Growth Factor (FGF), a portion of FGF, a nucleic acidencoding a polypeptide comprising at least a portion of an FGF, VascularEpithelial Growth Factor (VEGF), at least a portion of VEGF, a nucleicacid encoding a polypeptide comprising at least a portion of VEGF,Insulin-like Growth Factor-1 (IGF-1), at least a portion of IGF-1, anucleic acid encoding a polypeptide comprising at least a portion ofIGF-1, tumor amplified kinase STK15 (also BTAK and aurora2), a portionof STK15, a nucleic acid encoding a polypeptide comprising at least aportion of STK15, TMS-1, a portion of TMS-1, a nucleic acid encoding apolypeptide comprising at least a portion of TMS-1, maspin, at least aportion of maspin, a nucleic acid encoding a polypeptide comprising atleast a portion of maspin, at least a portion of breast cancerassociated (BRCA) gene, at least a portion of a BRCA gene product; CDw60protein, a portion of CDw60 protein or polypeptide, a nucleic acidencoding a polypeptide comprising at least a portion of CDw60 protein orpolypeptide, mammary expressed enzymes including cytochrome P450s,catechol-O-methyltransferase, epoxide hydrolase, peroxidases,glutathione S-transferases, N-acetyltransferases, or sulfotransferases,a nucleic acid encoding at least a portion of a mammary expressedenzyme, Kallikrein 6 (zyme/protease M/neurosin) protein or polypeptide(hK6), a nucleic acid encoding at least a portion of an hK6 protein orpolypeptide; and mammastatin protein or polypeptide, and a nucleic acidencoding at least a portion of a mammastatin protein or polypeptide. 35.A method to aid in diagnosing breast cancer or pre-cancer comprising:sequentially placing a ductal access tool comprising a single lumen inat least two breast ducts of a single breast of a patient, wherein thesingle lumen has an inner diameter large enough to retrieve clusters ofgreater than 10 cells; infusing a fluid into the duct through the singlelumen; and retrieving ductal fluid samples from each of the sequentiallyaccessed ducts through the single lumen, wherein the ductal fluidsamples comprise ductal epithelial cells and are not mixed with ductalfluid samples from any other ducts of the breast; and: examining theductal fluid samples to determine absorption of a molecule by abnormalcells in the fluid.
 36. A method as in claim 35, wherein the moleculecomprises iodide.
 37. A method as in claim 31, further comprising:examining the ductal fluid samples for a loss of heterozygosity.
 38. Amethod as in claim 31, further comprising examining the ductal fluidsamples for the presence of two or more markers.
 39. A method as inclaim 31, further comprising examining the ductal fluid samples for theabsence or two or mere markers.
 40. A method as in claim 31, furthercomprising examining the ductal fluid samples for the presence of atleast one marker and the absence of at least one marker.
 41. A method asin claim 31, further comprising analyzing collected ductal epithelialcells by cytology.
 42. A method as in claim 39, wherein the markers areselected from the group consisting of DNA content, p53 gene or geneproduct, and G-actin or a nucleic acid encoding a polypeptide comprisingat least a portion of G-actin.
 43. A method for analyzing breast markersor epithelial cells, comprising: sequentially placing a ductal accesstool comprising a single lumen in at least two breast ducts of a singlebreast of a patient, wherein the single lumen has an inner diameterlarge enough to retrieve clusters of greater than 10 cells; infusing afluid into the duct through the single lumen; and retrieving ductalfluid samples from each of the sequentially accessed ducts through thesingle lumen, wherein the ductal fluid samples comprise ductalepithelial cells and are not mixed with ductal fluid samples from anyother ducts of the breast.
 44. A method as in claim 43, wherein theducts from which the ductal fluid samples are retrieved are notspontaneously discharging ductal fluid.
 45. A method as in claim 43,wherein the marker is selected from the group consisting of:lysophosphatidic acid, a lysophospholipid, palladin, a portion ofpalladin, a nucleic acid encoding a polypeptide comprising at least aportion of palladin Lg, a portion of Lg, a nucleic acid encoding apolypeptide comprising at least a portion of Lg, E2F1, a portion ofE2F1, a nucleic acid encoding a polypeptide comprising at least aportion of E2F1, T1A12/mac 25, a portion of T1A12/mac 25, a nucleic acidencoding a polypeptide comprising at least a portion of T1A12/mac 25,MAGUK/ZO-1, a portion of MAGUK/ZO-1, a nucleic acid encoding apolypeptide comprising at least a portion of MAGUK/ZO-1, repressor ofestrogen receptor activity (REA), a portion of REA, a nucleic acidencoding a polypeptide comprising at least a portion of REA, prothymosinalpha (PTA), a portion of PTA, a nucleic acid encoding a polypeptidecomprising at least a portion of PTA, c-raf kinase, a portion of c-rafkinase, a nucleic acid encoding a polypeptide comprising at least aportion of c-raf kinase, CD66a, a portion of CD66a, a nucleic acidencoding a polypeptide comprising at least a portion of CD66a, KL-1, aportion of KL-1, a nucleic acid encoding a polypeptide comprising atleast a portion of KL-1, cell adhesion molecule 5.2 (CAM 5.2), a portionof CAM 5.2, a nucleic acid encoding a polypeptide comprising at least aportion of CAM 5.2, leptin, a portion of leptin, a nucleic acid encodinga polypeptide comprising at least a portion of leptin, Bcl-2 geneproduct, a portion of Bcl-2 gene product, a nucleic acid encoding apolypeptide comprising at least a portion of Bcl-2 gene product, nuclearmatrix 23 (nm23), a portion of nm23, a nucleic acid encoding apolypeptide comprising at least a portion of nm23, an apoptosis-relatedprotein, a portion of said protein, a nucleic acid encoding apolypeptide comprising at least a portion of the apoptosis-relatedprotein, lipocalin NGAL, a portion of lipocalin NGAL, a nucleic acidencoding a polypeptide comprising at least a portion of lipocalin NGAL,complement regulatory protein CD 46, a portion of CD 46, a nucleic acidencoding a polypeptide comprising at least a portion of CD 46,complement regulatory protein CD 59, a portion of CD 59, a nucleic acidencoding a polypeptide comprising at least a portion of CD 59, a nucleicacid encoding a portion of an FHIT gene, loss of heterozygosity at anFRA3B site, MRP-1/CD9, a portion of MRP-1/CD9, a nucleic acid encoding apolypeptide comprising at least a portion of MRP-1/CD9, KA11/CD82, aportion of KA11/CD82, a nucleic acid encoding a polypeptide comprisingat least a portion of KA11/CD82, a Fibroblast Growth Factor (FGF), aportion of FGF, a nucleic acid encoding a polypeptide comprising atleast a portion of an FGF, Vascular Epithelial Growth Factor (VEGF), atleast a portion of VEGF, a nucleic acid encoding a polypeptidecomprising at least a portion of VEGF, Insulin-like Growth Factor-1(IGF-1), at least a portion of IGF-1, a nucleic acid encoding apolypeptide comprising at least portion of IGF-1, tumor amplified kinaseSTK15 (also BTAK and aurora2), a portion of STK15, a nucleic acidencoding a polypeptide comprising at least a portion of STK15, TMS-1, aportion of TMS-1, a nucleic acid encoding a polypeptide comprising atleast a portion of TMS-1, maspin, at least a portion of maspin, anucleic acid encoding a polypeptide comprising at least a portion ofmaspin, at least a portion of breast cancer associated (BRCA) gene, andat least a portion of a BRCA gene product; CDw60 protein, a portion ofCDw60 protein or polypeptide, a nucleic acid encoding a polypeptidecomprising at least a portion of CDw60 protein or polypeptide, mammaryexpressed enzymes including cytochrome P450s,catechol-O-methyltransferase, epoxide hydrolase, peroxidases,glutathione S transferases, N-acetyltransferases, or sulfotransferases,a nucleic acid encoding at least a portion of a mammary expressedenzyme, Kallikrein 6 (zyme/protease M/neurosin) protein or polypeptide(hK6), a nucleic acid encoding at least a portion of an hK6 protein orpolypeptide; and mammastatin protein or polypeptide, and a nucleic acidencoding at least a portion of a mammastatin protein or polypeptide. 46.A method as in claim 43, wherein the marker is absorption of a moleculeby abnormal cells in the fluid.
 47. A method as in claim 46, wherein themolecule comprises iodide.
 48. A method as in claim 43, wherein themarker is a loss of heterozygosity.
 49. A method as in claim 43, whereinthe presence of two or more markers is determined.
 50. A method as inclaim 43, wherein the absence of two or more markers is determined. 51.A method as in claim 43 wherein the presence of at least one marker andthe absence of at least one marker is determined.
 52. A method as inclaim 43 wherein the marker determined is cytology of ductal epithelialcells.
 53. A method as in claim 43, wherein the marker is selected fromthe group consisting of DNA content, p53 gene or gene product, andG-actin or a nucleic acid encoding a polypeptide comprising at least aportion of G-actin.